Oxidative stress is a major contributor to aging, insulin resistance, and neurodegeneration. An emergent strategy for restoring redox homeostasis involves activation of the transcription factor, Nrf2 (nuclear factor erythroid 2-related factor 2), a member of the cap'n'collar family of basic leucine zipper transcription factors that regulates a coordinated adaptive gene program (MOI et al., Proc Natl Acad Sci USA, 91: 9926-9930 (1994)). Indeed, activators of the Nrf2 response are beneficial for the treatment and prevention of chronic degenerative diseases, while inhibitors of its activation may help to fight cancer (CALABRESE et al., Neurochem Res, 33: 2444-2471 (2008); HAYES et al., Trends Biochem Sci, 34, 176-188 (2009); LAU et al., Pharmacol Res, 58: 262-270 (2008)). A major challenge in the development of effective Nrf2 activators is to identify those that lead specifically to Nrf2 stabilization and consequent promoter activation, without imposing general oxidative/electrophilic stress.
Nrf2 is sequestered under homeostatic conditions by binding to its inhibitory protein, Keap1 (Kelch-like ECH-associated protein-1) (MOTOHASHI et al., Trends Mol Med, 10: 549-557 (2004); ITOH et al., Genes Dev 13: 76-86 (1999)). Keap1 serves as a bridge between Nrf2 and the Cul3-Rbx1 E3 ubiquitin ligase, leading to Nrf2 ubiquitination and thereby targeting Nrf2 for degradation by the 26S proteasome (KOBAYASHI et al., Mol Cell Biol, 24: 7130-7139 (2004); CULLINAN et al., Mol Cell Biol, 24: 8477-8486 (2004); ZHANG et al., Mol Cell Biol, 24: 10941-10953 (2004)). Upon exposure to oxidative stress, xenobiotics, or electrophilic compounds, the Nrf2 protein is released from its complex with Keap1 and translocates to the nucleus. There, it forms heterodimers with other transcription regulators, such as small Maf proteins, and induces the expression of antioxidant genes controlled by the antioxidant response element (ARE) (KASPAR et al., Free Radic Biol Med, 47: 1304-1309 (2009)).
Nrf2 is composed of Neh1-Neh6 domains, among which Neh2 is the putative negative regulatory domain that interacts with Keap1, Neh4 and Neh5 are transactivation domains, and Neh1 is the binding domain for ARE (TONG et al., Biol Chem, 387: 1311-1320 (2006b)). The functional domains of Keap1 are the Broad complex, Tramtrack and Bric-a-Brac (BTB), the intervening region (IVR), the double glycine repeats domain (DGR), and the C-terminal region (CTR) (TONG et al., Biol Chem, 387: 1311-1320 (2006b)). Two motifs in the Neh2 domain, e.g. ETGE and DLG, are recognized by the Keap1 homodimer in a hinge-latch mode (TONG et al., Mol Cell Biol, 26: 2887-2900 (2006a); TONG et al., Biol Chem, 387: 1311-1320 (2006b); TONG et al., Mol Cell Biol., 27: 7511-7521 (2007)). Keap1 mediates polyubiquitination of the positioned lysines within the central α-helix of the Neh2 domain under homeostatic conditions. Under oxidative/electrophilic stress reactive cysteines within Keap1 are modified and thus Keap1 undergoes conformational changes which lead to the detachment of the weak-binding DLG, resulting in Nrf2 stabilization. However, debate remains as to whether Nrf2 is completely released from its complex with Keap1 (ZHANG, Drug Metab Rev, 38: 769-789 (2006)) or not. Nrf2 activators identified so far are represented by potent alkylating agents (DINKOVA-KOSTOVA et al., Methods Enzymol, 382: 423-448 (2004)) and redox active compounds like diphenols, aminophenols and phenylene diamines, the precise mechanism of action of which is controversial. Recent data shows an enhanced effect of these compounds in the presence of exogenously added copper (WANG et al., Chem Biol, 17: 75-85 (2010)).
Current techniques for monitoring Nrf2 activation include the ARE-luciferase (MOEHLENKAMP et al., Arch Biochem Biophys, 363: 98-106 (1999)), Nrf2 responsive element-luciferase (Westerink et al., Mutat Res 696, 21-40 (2010)), or ARE-human placental alkaline phosphatase reporter systems (Son et al., J Neurochem 112, 1316-1326 (2010)).
Recently, a GFP fusion protein with the Nrf2 ZIP domain was utilized to study Nrf2 nuclear translocation (THEODORE et al., J Biol Chem, 283: 8984-8994 (2008)), while GFP fusion with the C. elegans Nrf2 analog was used to analyze Nrf2 activation by proteasomal dysfunction (KAHN et al., J. Biochem, 409: 205-213 (2008)). The ARE-GFP reporter assay was used to screen the library of 2,000 biologically active compounds (Spectrum library) and 45 hits identified (SHAW et al., UK Patent Application #0918626.3, Priority Date (Oct. 24, 2008), Publ Date (May 5, 2010)), with andrographolide being the most potent. The use of ARE-luciferase reporter for high throughput screening (HTS) purposes has been recently published (HUR et al., Chem Biol, 17, 537-547 (2010)). The screen of 1.5 million compounds resulted in discovery of novel alkylating agents targeting Cys 151 in Keap1 as well as a dozen other cellular proteins including phosphatase 2a, and HDAC1 and HDAC2 (HUR et al., Chem Biol, 17, 537-547 (2010)).
ITOH et al., Genes Dev 13: 76-86 (1999) disclosed a NEH2+ reporter construct, and used it to assay NRF2 activity. This paper describes a chicken Neh2 construct, and a mouse Neh2 construct. The latter is 1-73 aa residues of mouse Neh2 attached to GFP. As shown in FIG. 9 of that paper, the construct provides a very strong fluorescent signal, indicating that it is poorly recognized by endogenous Keap1 and therefore accumulates in the cell. The construct is not applicable for HTS purposes. Itoh et al. demonstrate that their fusion is ubiquitated (prepared for destruction) but they do not show that it is degraded. The reporter in Itoh et al is attached to the N terminus.